A New Co-Expressed P450 and UGT Platform for the Simultaneous Preparation of P450 Metabolites and Their Glucuronides

用于同时制备 P450 代谢物及其葡萄糖苷酸的新型共表达 P450 和 UGT 平台

• 批准号: 9345284
• 负责人: Enrique Martinez
• 金额: $ 21.44万
• 依托单位: OXFORD BIOMEDICAL RESEARCH, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-20 至 2018-09-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9345284
• 关键词: 4-methylumbelliferone; Agriculture; Artificial Liver; Bile fluid; Biomedical Research; Buffers; CYP3A4 gene; Catalysis; Cell Wall; Chemicals; Cytochrome P450; Cytochromes; Detergents; Development; Drug Industry; Enzymes; Exhibits; Genes; Glucose; Glucuronides; Glucuronosyltransferase; Glycosides; Goals; Hepatic; Hour; Human; In Situ; Liver; Liver Extract; Membrane; Metabolic; Metabolism; Methods; Microsomes; Midazolam; NADP; Natural regeneration; Organic solvent product; Oxidoreductase; Pharmacologic Substance; Phase; Physiological; Pichia; Powder dose form; Preparation; Process; Production; Property; Protein Isoforms; Reaction; Recombinants; Reporting; Research; Research Personnel; Solubility; System; Technology; Temperature; Time; Tissues; Toxin; UGT2B7 UDP-glucuronosyltransferase; Uridine Diphosphate; Uridine Diphosphate Glucose Dehydrogenase; Uridine Diphosphate Glucuronic Acid; Urine; Work; Xenobiotic Metabolism; Xenobiotics; Yeasts; base; catalyst; cofactor; cost; cost effectiveness; drug candidate; gene cloning; improved; in vivo; innovation; macromolecule; milligram; novel; overexpression; oxidation; protein protein interaction; screening; tool

Toxins, pharmaceuticals and physiologically indogenous compounds are metabolized by the liver via several classes of enzymes. The uridine diphosphate glucuronosyltransferases (UGTs) are concentrated in the liver but also present in other tissues. These enzymes add UDP-glucuronic acid (UDPGA) to a compound, thereby increasing its solubility and ability to be excreted in the bile and urine. Oxford Biomedical Research has created a proprietary method for processing a strain of Pichia pastoris optimized for expressing the cytochromes P450 (CYP) and oxidoreductase, resulting in a growing product line tailored to pharmaceutical research. This process transforms the yeast into a stabilized dry powder (SDP) format, which exhibits remarkable organic solvent and thermal tolerance along with extended catalysis when compared to traditional recombinant enzymes. The proposed multi-catalytic platform, capable of simultaneous P450 oxidation followed by UGT glucuronidation, would give the pharmaceutical researcher a valuable tool for preparing glucuronides as well as studying the P450/UGT synergistic relationship. This Phase I proposal will involve producing and optimizing three catalytic systems:the first Pichia system will express the dehydrogenase responsible for converting UDP-glucose to UDPGA, the cofactor required for glucuronide production. The second system will include expand on this platform and include the UGT2B7 enzyme to create a catalyst capable of glucuronidating 4-methylumbelliferone. The third and final system will add into this expression system the CYP3A4 and P450 oxidoreductase (POR) enzymes, with development focusing on the optimization of 1'-hydroxymidazolam glucuronide production. Achieving these goals will give pharmaceutical research a robust, easy-to-use system for performing two metabolically-important reactions in one system; P450 oxidation followed by UGT glucuronidation.

毒素、药物和生理学内源性化合物由肝脏通过多种途径代谢 酶的类别。尿苷二磷酸葡萄糖醛酸基转移酶 (UGT) 集中在 肝脏，但也存在于其他组织中。这些酶将 UDP-葡萄糖醛酸 (UDPGA) 添加到化合物中， 从而增加其溶解度和在胆汁和尿液中排泄的能力。 牛津生物医学研究中心创造了一种处理毕赤酵母菌株的专有方法 针对细胞色素 P450 (CYP) 和氧化还原酶的表达进行了优化，从而扩大了产品线 专为药物研究而定制。该过程将酵母转化为稳定的干粉 (SDP) 格式，表现出显着的有机溶剂和耐热性以及扩展的催化作用 与传统的重组酶相比。所提出的多催化平台能够同时 P450 氧化后进行 UGT 葡萄糖醛酸化，将为药物研究人员提供一个有价值的工具 制备葡萄糖苷酸以及研究P450/UGT协同关系。 该第一阶段提案将涉及生产和优化三个催化系统：第一个毕赤系统将 表达负责将 UDP-葡萄糖转化为 UDPGA 的脱氢酶，UDPGA 是 葡萄糖苷酸生产。第二个系统将包括在此平台上的扩展并包括 UGT2B7 酶产生能够使 4-甲基伞形酮葡萄糖醛酸化的催化剂。第三个也是最后一个系统 将在该表达系统中添加 CYP3A4 和 P450 氧化还原酶 (POR) 酶， 开发重点是优化 1'-羟基咪达唑仑葡萄糖醛酸生产。 实现这些目标将为药物研究提供一个强大、易于使用的系统，用于执行两个任务 一个系统中重要的代谢反应； P450 氧化，随后 UGT 葡萄糖醛酸化。